Summary The regulation of gene expression during embryonic development in Drosophila melanogaster, as in most other organisms, is highly dependent on the activity of cis-regulatory modules (CRMs) in the genome. Transcription factor (TF) proteins bind to such sequences, regulating expression of target genes by either activating or repressing transcription. A critical question in modern developmental biology is how the architecture of multiple functional TF binding sites is organized in CRMs to control the regulatory output. TFs frequently bind many sites within the genome with varying affinity and are capable of complex protein-protein interactions. The goal of this project is use integrated interdisciplinary, systems-level experimental, computational, bioinformatic and mathematical approaches to investigate the regulatory logic that controls the activity of TF binding and interactions at CRMs and the impact on gene expression in early Drosophila development. The results will inform our fundamental understanding of key transcriptional regulatory mechanisms during embryonic development. In the early Drosophila embryo, CRMs, under the control of TF concentration gradients, direct the spatio-temporal expression of a network of developmental genes, including the highly conserved homeotic genes in the bithorax complex (BX-C). The homeotic genes are critical for differentiation and specification of many important structures in development. Preliminary studies from the PIs? labs show that CRM sequences in the BX-C are rapidly evolving in Drosophila species. Despite the general lack of conservation, these experiments discovered the preservation of an organized spatial arrangement of critical TF binding sites, representing signature motifs, within a number of CRMs. The ability to utilize integrated experimental approaches provides an exciting opportunity to expand the scope of the functional analysis of these signature motifs in CRMs. The ultimate goal of this proposal is to elucidate the molecular mechanisms that control functional activity of homeotic gene CRMs at the sequence level. The long term scientific goal of the PIs is to fully investigate the mechanisms by which genomic regulatory regions achieve coordinated control of gene expression in the early developmental networks in Drosophila. The major focus of this proposal will be to apply a systems-level approach to decipher the precise molecular control of TF binding and interactions within CRMs in the BX-C of Drosophila melanogaster. The discoveries from preliminary studies will be used as a foundation to specifically address the molecular contribution of individual TF binding sites within known CRMs, investigate their combinatorial interactions, and analyze their in vivo activity in two complementary Aims that incorporate both experimental and computational approaches. Specifically, the proposed studies will address the mechanisms at play in the CRMs, including the functional role of binding site affinity, the specificity and distance-dependence of interactions between repressor and activator TFs, and the role for distance-dependent cooperativity between activator proteins. Undergraduate students will be critical to the success of this project. Indeed, the contribution from students will be essential to the completion of the proposed research and as a result they will receive extensive guidance and share authorship on publications. Clark University has a strong tradition of excellence in undergraduate research. Both PIs have made substantial efforts to integrate their teaching and research at a very high level. Since Dr. Dresch?s Ph.D. in 2012, she has worked with 28 undergraduate students on interdisciplinary, collaborative research projects. She has published with 22 undergraduate co-authors, representing 20 individual students. During the past fifteen years, Dr. Drewell has mentored 12 graduate students and 104 undergraduate students in his research lab. He has published with 61 undergraduate co-authors, representing 35 individual students. This funding will ensure the continuation of this level of student involvement in both laboratories. Dr. Dresch and Dr. Drewell will also continue in their leadership roles in the recently established Mathematical Biology and Bioinformatics Concentration at Clark University. The PIs will continue to develop an expanded curriculum for the Concentration, including refining their new cross-departmental course in Mathematical Modeling of Biological Systems, which focuses on the dynamic nature and power of interdisciplinary approaches in life science. Dr. Drewell and Dr. Dresch are also actively involved in the newly established SACNAS student chapter at Clark University which will support students from traditionally under-represented groups in STEM fields and Dr. Dresch is the faculty mentor for the Clark Women in STEM group.